Functionally integrated palindromic hairpin-enabled signal efficient amplification and continuous transduction for one-pot miRNA sensing.

In this work, we developed an ultrasensitive method for microRNA (miRNA) sensing based on palindrome-mediated isothermal cascade nicking/polymerization for DNA amplification and fluorescent copper nanoparticle (CuNP) generation for signal transduction. With rational palindrome design, only two DNA hairpins and a pair of enzymes were needed to realize one-pot and two-step miRNA detection. In the first step, the target miRNA unfolded the nicking site-containing RNA Probe that subsequently hybridized with the palindromic sequence-engineered hairpin and initiated multiple isothermal cycles driven by polymerase and endonuclease to complete target recognition and signal amplification. Then, the abundant DNA templates accumulated in the previous step facilely and rapidly guided the in situ generation of fluorescent CuNPs for signal transduction. The efficient isothermal amplification and effortless signal transduction jointly achieved ultrasensitive miRNA-21 sensing with a low detection limit (9.7 fM) in a simple and convenient manner. In addition, thanks to its high selectivity and anti-interference ability, the method was able to unambiguously distinguish cancer cells from normal cells based on the test results of cellular miRNA-21. Moreover, this method also enables the detection of different miRNAs simply by modifying the probe sequence, which demonstrates high sensing versatility and application potential in advanced molecular diagnostics.

Salmonellosis remains one of the leading causes of bacterial gastrointestinal infections in humans and animals. Molecular diagnostics has dramatically reshaped the diagnostic landscape for Salmonella infection; however, it remains time- and resource-intensive. Isothermal DNA amplification, for example loop isothermal amplification (LAMP), performed at a constant temperature, is the basis for the development of rapid diagnostic tests that can be adapted to the point-of-care (PoC) formats and implemented in resource-limited settings or remote from centralized laboratories. The aim of this study was to develop and validate a novel LAMP-based method for detecting Salmonella enterica in human stool samples, wherein amplification results are monitored using a loop primer labeled with a fluorophore and an internal quencher. The proposed method achieves a limit of detection (LoD95) of 250 copies per reaction, with a sensitivity of 86.84% (95% CI: 71.91–95.59%) and specificity of 96.49% (95% CI: 87.89–99.57%) relative to qPCR, and demonstrates increased robustness against DNA amplification inhibitors present in fecal samples. Incorporation of distinct fluorophores into loop primers for FLP-LAMP targeting different genes could potentially enable multiplexing and simultaneous detection of multiple pathogens, thereby expanding the diagnostic utility of isothermal amplification.

Infectious diseases remain one of the major causes of death worldwide in developing countries. While screening via conventional polymerase chain reaction (PCR) is the gold standard in laboratory testing, its limited applications at the point-of-care have prompted the development of more portable nucleic acid detection systems. These include isothermal DNA amplification techniques, which are less equipment-intensive than PCR. Unfortunately, these techniques still require extensive sample preparation, limiting user accessibility. In this study, we introduce a novel system that combines thermophilic helicase-dependent amplification (tHDA) with a Triton X-100 micellar aqueous two-phase system (ATPS) to achieve cell lysis, lysate processing, and enhanced nucleic acid amplification in a simple, one-step process. The combined one-pot system was able to amplify and detect a target gene from whole-cell samples containing as low as 102cfu/mL, and is the first known application of ATPSs to isothermal DNA amplification. This system's ease-of-use and sensitivity underlie its potential as a point-of-care diagnostic platform to detect for infectious diseases. Graphical abstract ᅟ.

Double-stranded DNAs (ds-DNAs) have been identified as efficient templates favoring the formation of fluorescent copper nanoparticles (Cu NPs). Herein, we have tried to synthesize fluorescent Cu NPs using single-stranded DNAs (ss-DNAs) as templates and to identify the critical DNA sequences. By comparing the results using homopolymer DNAs, hairpin DNAs, and pristine ss-DNAs as templates, we found that DNA thymine base plays a dominant role in producing red-emissive fluorescent Cu NPs on ss-DNA templates. The thymine-dependent growth of the fluorescent Cu NPs is confirmed by Hg2+ mediated T–T base pair in comparison with the other non-specific metal ions, which could be developed into a practical sensor for turn-on fluorescence detection of Hg2+ with a high selectivity. The mechanism is briefly discussed according the DNA sequence-dependent formation of fluorescent Cu NPs. This work demonstrates the sequence role in producing fluorescent Cu NPs that could serve as promising fluorescent nanoprobes in biosensing and DNA-hosted Cu nanomaterials.

In situ formation of fluorescent copper nanoparticles for ultrafast zero-background Cu2+ detection and its toxicides screening

Isothermal DNA amplification combined with lateral flow dipsticks for detection of biothreat agents

Guanine-rich sequences are able to form quadruplexes consisting of G-quartet structural units. Quadruplexes play an important role in the regulation of gene expression and have therapeutic and biotechnological potential. The HIV-1 integrase inhibitor, (GGGT)4 , and its variants demonstrate unusually high thermal stability. This property has been exploited in the use of quadruplex formation to drive various endergonic reactions of nucleic acids such as isothermal DNA amplification. Quadruplex stability is mainly determined by cations, which specifically bind into the inner core of the structure. In the present work, we report a systematic study of a variant of the HIV-1 integrase inhibitor, GGGTGGGTGGGTGGG (G3T), in the presence of alkali and alkaline-earth cations. We show that Sr(2+) -G3T is characterized by the highest thermal stability and that quadruplex formation requires only one Sr(2+) ion that binds with low micromolar affinity. These concentrations are sufficient to drive robust isothermal quadruplex priming DNA amplification reaction. The Sr(2+) -quadruplexes are also able to form unusually stable dimers through end-to-end stacking. The multimerization can be induced by a combination of quadruplex forming cations (i.e., K(+) or Sr(2+) ) and non-specific Mg(2+) .

When imaging cells, nuclear counterstaining is imperative; however, many commercial nuclear-staining dyes based on nucleic acid intercalation result in nonspecific signals in the cytoplasm. Here, we propose a new strategy that stains the nucleus with high specificity by in situ formation of DNA-templated copper nanoparticles (CuNPs). We demonstrated that genomic DNA in the nucleus enabled rapid formation of highly fluorescent CuNPs immediately following addition of a copper ion source and ascorbate as a reducing agent. Moreover, we found that RNA and mitochondrial DNA, largely responsible for nonspecific cytoplasmic signals from commercial nuclear-staining dyes, did not mediate the formation of the highly fluorescent CuNPs, resulting in highly specific nuclear staining at a reduced cost relative to commercially available methods. Furthermore, we verified the compatibility of the proposed method with other fluorescence-labeling techniques. These results demonstrated the efficacy of this method and its promise as a powerful tool for cell imaging.

Ion sensing (EIS) real-time quantitative monitorization of isothermal DNA amplification

Isothermal DNA amplification is an alternative to PCR-based amplification for point-of-care diagnosis. Since the early 1990s, the approach has been refined into a simple, rapid and cost-effective tool by means of several distinct strategies. Input signals have been diversified from DNA to RNA, protein or small organic molecules by translating these signals into input DNA before amplification, thus allowing assays on various classes of biomolecules. In situ detection of single biomolecules has been achieved using an isothermal method, leveraging localized signal amplification in an intact specimen. A few pioneering studies to develop a homogenous isothermal protein assay have successfully translated structure-switching of a probe upon target binding into input DNA for isothermal amplification. In addition to the detection of specific targets, isothermal methods have made whole-genome amplification of single cells possible owing to the unbiased, linear nature of the amplification process as well as the large size of amplified products given by ϕ29 DNA polymerase. These applications have been devised with the four isothermal amplification strategies covered in this review: strand-displacement amplification, rolling circle amplification, helicase-dependent amplification and recombinase polymerase amplification.

CRISPR-Cas system has evolved as a highly preferred genetic engineering tool to perform target gene manipulation via alteration of the guide RNA (gRNA) sequence. The ability to recognize and cleave a specific target with high precision has led to its applicability in multiple frontiers pertaining to human health and medicine. From basic research focused on understanding the molecular basis of disease to translational approach leading to early and precise disease diagnosis as well as developing effective therapeutics, the CRISPR-Cas system has proved to be a quite versatile tool. The coupling of CRISPR-Cas mediated cleavage with isothermal amplification (ISA) of target DNA, followed by a read-out using fluorescent or colorimetric reporters appears quite promising in providing a solution to the urgent need for nucleic acid-based point-of-care diagnostic. Hence, it has been recognized as a highly sophisticated molecular diagnostic tool for the detection of disease-specific biomarkers not limited to nucleic acids-based detection but also of non-nucleic acid targets such as proteins, exosomes, and other small molecules. In this review, we have presented salient features and principles of class 2 type II, V, and VI CRISPR-Cas systems represented by Cas9, Cas12, and Cas13 endonucleases which are frequently used in molecular diagnosis. The article then highlights different medical diagnostic applications of CRISPR-Cas systems focusing on the diagnosis of SARS-CoV-2, Dengue, Mycobacterium tuberculosis, and Listeria monocytogenes. Lastly, we discuss existing obstacles and potential future pathways concerning this subject in a concise manner.

Since the development of polymerase chain reaction, amplification of nucleic acids has emerged as an elemental tool for molecular biology, genomics, and biotechnology. Amplification methods often use temperature cycling to exponentially amplify nucleic acids; however, isothermal amplification methods have also been developed, which do not require heating the double-stranded nucleic acid to dissociate the synthesized products from templates. Among the several methods used for isothermal DNA amplification, the helicase-dependent amplification (HDA) is discussed in this review with an emphasis on the reconstituted DNA replication system. Since DNA helicase can unwind the double-stranded DNA without the need for heating, the HDA system provides a very useful tool to amplify DNA in vitro under isothermal conditions with a simplified reaction scheme. This review describes components and detailed aspects of current HDA systems using Escherichia coli UvrD helicase and T7 bacteriophage gp4 helicase with consideration of the processivity and efficiency of DNA amplification.

Towards on-site detection of gluten-containing cereals with a portable and miniaturized prototype combining isothermal DNA amplification and naked eye detection

Rolling circle amplification (RCA) developed in the mid-1990s has been widely used as an efficient isothermal DNA amplification process for molecular diagnosis. This enzymatic process amplifies target DNA sequences with high fidelity and specificity by using the strand displacing DNA polymerases. The product of RCA is long single-stranded DNA that contains tandem repeat of target sequence. Isothermal reaction amplification condition of RCA has an advantage over conventional polymerase chain reaction, because no temperature cycling devices are needed for RCA. Thus, RCA is suitable tool for point-of-care detection of target nucleic acids as well as facile detection of target genes. Combined with various detection methods, RCA could amplify and detect femtomolar scale of target nucleic acids with a specificity of one or two base discrimination. Herein, RCA technology is reviewed with an emphasis on molecular diagnosis of microRNAs, infectious pathogens, and point mutations.

Microfluidic droplet generation affords precise, low volume, high throughput opportunities for molecular diagnostics. Isothermal DNA amplification with bioluminescent detection is a fast, low-cost, highly specific molecular diagnostic technique that is triggerable by temperature. Combining loop-mediated isothermal nucleic acid amplification (LAMP) and bioluminescent assay in real time (BART), with droplet microfluidics, should enable high-throughput, low copy, sequence-specific DNA detection by simple light emission. Stable, uniform LAMP–BART droplets are generated with low cost equipment. The composition and scale of these droplets are controllable and the bioluminescent output during DNA amplification can be imaged and quantified. Furthermore these droplets are readily incorporated into encapsulated droplet interface bilayers (eDIBs), or artificial cells, and the bioluminescence tracked in real time for accurate quantification off chip. Microfluidic LAMP–BART droplets with high stability and uniformity of scale coupled with high throughput and low cost generation are suited to digital DNA quantification at low template concentrations and volumes, where multiple measurement partitions are required. The triggerable reaction in the core of eDIBs can be used to study the interrelationship of the droplets with the environment and also used for more complex chemical processing via a self-contained network of droplets, paving the way for smart soft-matter diagnostics.